5' RACE

How did you clean your 1st strand cDNA before tailing? It is important to
remove the digested RNA and primers before doing TdT tailing, according to
the kit instructions. I used the same kit, and always got something after
second round of PCR using a nested primer. The product in the 1st RACE PCR
is usually invisible in gel. I use GSP1 for RT, GSP2 and acchor primer for
RACE pcr. You need re-PCR using 1uL of neat or 1/20 diluted (I would do
both) RACE PCR reaction as template, and primers GSP3 and the adaptor
primer (AUAP?).
Hope this of help.
>I have been trying to amplify the 5' end of a particular cDNA using 5'
>RACE. I've been using the GIBCO/BRL 5' RACE kit. I know that I have
>synthesized intact cDNA, since I can easily amplify it using internal
>primers (a nice fat product easily visible on a gel). I believe my
>TdT is OK, since I have used it to end label double-stranded DNA with
>3' protruding ends, and I can amplify a fragment from that end-labeled
>double-stranded DNA using the same anchor primer and gene-specific
>primer(s) that I have tried using with 5'RACE. I have tried 4
>different gene-specific primers (none of which should amplify a
>product over 500 bp), as well as nested amplification, with no
>results. I think my problem lies with the TdT step, but I can't think
>what the problem might be, since the enzyme works fine with my control
>DNA. Has anyone else experienced this problem? I would appreciate
>any suggestions or advice.
>>I've also tried ligation-anchored PCR, but with no luck there either.
>Has anyone had any success with this method?
>>Thanks,
>>Gae Kovalick
>Department of Zoology
>Miami University
>Oxford, OH 45056
>>kovalige at muohio.edu
ZhongLin Chai, PhD
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Department of Pathology and Immunology
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